Modulation neuroinflammation through interference of cooperative microRNA-RNA-binding protein interactions

Yale University, New Haven CT

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Abstract

The importance of Th17 cells, and their potent inflammatory cytokines (IL-17A and GM-CSF), in multiple sclerosis (MS) and other autoimmune diseases is established. In animal MS models, Th17 cells are recruited and localized to the CNS through ?2 integrin LFA-1-dependent adhesion and transendothelial migration. Although transcriptional regulation of the IL-17A and GM-CSF genes has been well characterized, the mRNAs encoding these cytokines are highly labile and must be dynamically regulated to allow significant gene expression. We have demonstrated that T cell adhesion through ?2 integrin engagement results in marked stabilization of mRNAs encoding TNF-? and IFN-?, through modulation and nuclear-to-cytosolic translocation of the RNA-binding protein (RBP) HuR. Our preliminary data support an equally remarkable extension of the IL-17A and GM-CSF transcript half-lives through an LFA-stimulated, HuR-dependent mechanism. When attempting to characterize potential competitive microRNA (miRNA)- HuR interactions on the IL-17A 3'- untranslated region (3'-UTR), we unexpectedly detected a cooperative, interdependent RNA- stabilizing interaction between miR-466l-3p and HuR. We mapped the miR-466l-3p target site within the IL-17A 3'-UTR. An oligonucleotide preventing this interaction (target site blocker [TSB]) inhibits LFA-1-induced, HuR- dependent IL-17A mRNA stabilization, and enhanced IL-17A production, in a cytokine-specific manner. We intend to define the same for GM-CSF, as its mRNA's 3'-UTR contains a highly conserved AU-rich element which includes 4 potential miR-466l-3p target sites. Our previously published and new data, and the pathogenic importance of IL-17A and GM-CSF in neuroinflammation, have led to our hypothesis, that leukocyte integrin engagement promotes Th17 cell IL-17A and GM-CSF expression via enhanced cooperative binding of HuR and miR-466l-3p to their 3'-UTRs, and that this potent pro-inflammatory switch is amenable to novel therapeutic targeting. Specific proposals now are to: (1) map the miR-466l-3p target site in the GM-CSF 3'-UTR, and generate an effective, specific TSB, using complementary molecular approaches including (a) MS2-TRAP 3'-UTR/miRNA pulldowns, and (b) pBBB ? globin RNA reporter stability assays; and (2) determine the impact of selectively blocking miR-466l-3p's interaction with the IL-17A and GM- CSF transcripts on immunopathology in a chronic, myelin oligodendrocyte glycoprotein (MOG)-specific 2D2 transgenic EAE model, and a relapsing, remitting, proteolipid protein peptide (PLP)-immunized EAE model, evaluating EAE clinical scores, as well as CNS IL-17A and GM-CSF mRNA and protein levels. The novelty of this newly described cooperative miRNA-RBP interaction, and our ability to test inhibitors directed at this cooperativity in neuroinflammation disease models, makes this both a molecular and a highly translational exploratory R21 project. We hope this work directs more extensive efforts in posttranscriptional regulation of pathogenic cytokine expression, and defines a novel therapeutic targeting opportunity.